Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.
Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.
复制标题
使用毛细管电泳和荧光标记肽检测感染传染性海绵状脑病的动物血液中的异常朊病毒蛋白。
DOI：
10.1016/s0021-9673(99)00514-2
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Kathryn R. Goodwin
中科院分区：
文献类型：
--
作者：
M. J. Schmerr;Allen L. Jenny;Marie S Bulgin;Janice M. Miller;Amirali N Hamir;Randall C. Cutlip;Kathryn R. Goodwin
人类和动物的传染性海绵状脑病是潜伏期长的致命性神经退行性疾病。这些疾病的假定原因是一种正常的宿主蛋白质，朊病毒蛋白质，变得改变。这种异常的朊病毒蛋白主要存在于疾病后期受感染个体的大脑中，但也可以在淋巴和其他组织中以较低的量发现。为了在动物中根除这种疾病，重要的是开发一种可以浓缩异常朊病毒蛋白的系统和一种非常灵敏的检测方法。毛细管电泳所能达到的灵敏度使得检测血液中的异常蛋白质成为可能。在氨基末端的肽合成过程中，用荧光素标记羧基末端区域的肽，氨基酸位置218-232。亲和纯化针对该肽产生的抗体，并用于毛细管电泳免疫测定。毛细管中荧光素标记的肽的量为50阿莫尔。血液取自正常绵羊和麋鹿、羊瘙痒病感染绵羊和慢性消耗性疾病感染麋鹿。通过常规方法制备血沉棕黄层和血浆。在用蛋白酶K处理后，其破坏正常蛋白质但不破坏改变的蛋白质，提取血液组分并在毛细管电泳免疫测定中检测异常朊病毒蛋白。在感染动物的血液中检测到异常朊病毒蛋白，但在正常动物的血液中没有检测到。该测定使得这些疾病的临床前测定成为可能，并且可以适用于测试用于制造供人类消费的药物和产品的工艺材料中的异常朊病毒蛋白。
Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218–232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.
影响因子：
7.4
作者：
SCHULTZ, NM;HUANG, L;KENNEDY, RT
通讯作者：
KENNEDY, RT